Excitatory sympathetic (spinal) autonomic reflexes from the heart regulate the cardiovascular system during myocardial ischemia and reperfusion. Cardiac sympathetic afferent stimulation leads to angina pectoris and life threatening hemodynamic adjustments, including hypertension and arrhythmias that both initiate and exacerbate ischemia and infarction. In the last grant cycle we evaluated afferent mechanisms involving chemical mediators including bradykinin (BK), serotonin (5HT) and histamine released during ischemia that play an important role in activating cardiac spinal nerve endings. We have identified cellular and metabolic sources of these mediators and have begun to look at interactions and neuronal signaling mechanisms. It is clear that other mediators are involved since inhibition of the action or synthesis of individual metabolites does not fully prevent afferent activation during ischemia. Recently, we identified two novel mediators, endothelin (ET) and thromboxane A2 (TxA2) that appear to serve a central role during myocardial ischemia by acting both independently and interactively with other mediators to stimulate ventricular nerve endings. We also have begun to study glutamate and nitric oxide (NO) and their interaction in the rostral ventral lateral medulla (rVLM), which processes cardiac spinal afferent information and regulates sympathetic outflow. We propose four sets of hypotheses on mechanisms of activation of cardiac afferents by: 1) TxA2 and its signaling through protein kinase C, 2) ET and the interactive role of ETA and ETB receptor stimulation, 3) interactions between TxA2, 5HT and BK through reciprocal actions on mediator production/release from platelets (TxA2-5HT) or the cyclooxygenase pathway (TxA2-BK), and 4) neurotransmitter roles and interaction between glutamate, its ionotropic receptors, and nitric oxide (NO) formed from neuronal, endothelial or inducible NO synthase in rVLM processing of cardiac afferent information using whole animal reflex, cellular electrophysiological and pharmacological approaches. New knowledge gained from these studies will allow a better understanding of mechanisms underlying the genesis of angina pectoris and reflex activation of the cardiovascular system during myocardial ischemia and reperfusion and may suggest therapeutic options directed at limiting morbidity and mortality associated with this common condition.